Sperm-Specific CatSper is Not Conserved in All Vertebrates and May Not be the Only Progesterone-Responsive Ion Channel Present in Sperm

Progesterone (P4) acts as a key conserved signalling molecule in vertebrate reproduction. P4 is especially important for mature sperm physiology and subsequent reproductive success. “CatSpermasome”, a multi-unit molecular complex, has been suggested to be the main if not the only P4-responsive atypical Ca2+-ion channel present in mature sperm. Altogether, here we analyse the protein sequences of CatSper1-4 from more than 500 vertebrates ranging from early fishes to humans. CatSper1 becomes longer in mammals due to sequence gain mainly at the N-terminus. Overall the conservation of full-length CatSper1-4 as well as the individual TM regions remain low. The lipid-water-interface residues (i.e. a 5 amino acid stretch sequence present on both sides of each TM region) also remain highly diverged. No specific patterns of amino acid distributions were observed. The total frequency of positively charged, negatively charged or their ratios do not follow in any specific pattern. Similarly, the frequency of total hydrophobic, total hydrophilic residues or even their ratios remain random and do not follow any specific pattern. We noted that the CatSper1-4 genes are missing in amphibians and the CatSper1 gene is missing in birds. The high variability of CatSper1-4 and gene-loss in certain clades indicate that the “CatSpermasome” is not the only P4-responsive ion channel. Data indicate that the molecular evolution of CatSper is mostly guided by diverse hydrophobic ligands rather than only P4. The comparative data also suggest possibilities of other Ca2+-channel/s in vertebrate sperm that can also respond to P4. Graphical Abstract In birds, only CatSper1 is absent. Similarly, in amphibians, all the CatSper units are absent, suggesting the presence of other proteins that can act as P4-responsive Ca2+-ion channels there. TRPV4 is present in all these vertebrate groups and multiple copies of the TRPV4 gene are present in amphibians Supplementary Information The online version contains supplementary material available at 10.1007/s00232-024-00316-1.

The transmembrane (TM) regions of all CatSperα were predicted using the TMHMM, Deep TMHMM, and TMSEG software.Some of these prediction-based TM regions differ by more than 5 amino acids than the experimentally characterized structure available in Uniport ID-Q8NEC5 for Catsper1, ID-Q96P56 for Catsper2, ID-Q86XQ3 for Catsper3, and ID-Q7RTX7 for Catsper4.Also, some of the transmembrane regions were not predicted at all by this software.Due to this variability, the human sequence from UniProt was used as the reference and all the transmembrane regions were identified across all the species for every analysis described in this paper.

Figure S1 :
Figure S1: The LWI residues of CatSper1 are not conserved.The Frequencies of individual amino acids present in the LWI region of different phyla are shown.Amino acids at the LWI regions of all 98 species including fishes (F), amphibians (A), reptiles (R), birds (B), and mammals (M) were shown in violet, blue, yellow, green, and red respectively.Their frequency of occurrence was plotted as Inner LWI (for the intracellular region, rightmost side), Outer LWI (for the extracellular region, middle), and Total LWI (left-most side).The amino acids were arranged in increasing order of their ΔG values of side chains.Their pKa values and hydrophobicity were shown on the right side.Amino acids snorkeling propensity was depicted as (***) high, (**) medium, (*) low, and (#) not at all on the right side.The dotted blue line in the individual graph depicts the natural abundance of individual amino acids in nature.

Figure S2 :
Figure S2: The LWI residues of CatSper2 are not conserved.The Frequencies of individual amino acids present in the LWI region of different phyla are shown.Amino acids at the LWI regions of all 138 species including fishes (F), amphibians (A), reptiles (R), birds (B), and mammals (M) were shown in violet, blue, yellow, green, and red respectively.Their frequency of occurrence was plotted as Inner LWI (for the intracellular region, rightmost side), Outer LWI (for the extracellular region, middle), and Total LWI (left-most side).The amino acids were arranged in increasing order of their ΔG values of side chains.Their pKa values and hydrophobicity were shown on the right side.Amino acids snorkeling propensity was depicted as (***) high, (**) medium, (*) low, and (#) not at all on the right side.The dotted blue line in the individual graph depicts the natural abundance of individual amino acids in nature.

Figure S3 :
Figure S3: The LWI residues of CatSper3 are not conserved.The Frequencies of individual amino acids present in the LWI region of different phyla are shown.Amino acids at the LWI regions of all 150 species including fishes (F), amphibians (A), reptiles (R), birds (B), and mammals (M) were shown in violet, blue, yellow, green, and red respectively.Their frequency of occurrence was plotted as Inner LWI (for the intracellular region, rightmost side), Outer LWI (for the extracellular region, middle), and Total LWI (left-most side).The amino acids were arranged in increasing order of their ΔG values of side chains.Their pKa values and hydrophobicity were shown on the right side.Amino acids snorkeling propensity was depicted as (***) high, (**) medium, (*) low, and (#) not at all on the right side.The dotted blue line in the individual graph depicts the natural abundance of individual amino acids in nature.

Figure S4 :
Figure S4: The LWI residues of CatSper4 are not conserved.The Frequencies of individual amino acids present in the LWI region of different phyla are shown.Amino acids at the LWI regions of all 137 species including fishes (F), amphibians (A), reptiles (R), birds (B), and mammals (M) were shown in violet, blue, yellow, green, and red respectively.Their frequency of occurrence was plotted as Inner LWI (for the intracellular region, rightmost side), Outer LWI (for the extracellular region, middle), and Total LWI (left-most side).The amino acids were arranged in increasing order of their ΔG values of side chains.Their pKa values and hydrophobicity were shown on the right side.Amino acids snorkeling propensity was depicted as (***) high, (**) medium, (*) low, and (#) not at all on the right side.The dotted blue line in the individual graph depicts the natural abundance of individual amino acids in nature.

table 1 :
List of all the sequences from different species used in this study.